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Department of Pathology (J.R.A.S., S.K.S., A.M.S.), University of Cambridge, Cambridge CB2 1QP; Department of Obstetrics and Gynaecology (J.R.A.S., S.K.S.,), University of Cambridge, The Rosie Hospital, Cambridge CB2 2SW; Microarray Development Group (T.C.F., R.J.S.), United Kingdom Human Genome Mapping Project Resource Centre, Cambridge CB10 1SB; School of Biological Sciences (S.K.), University of Manchester, Manchester M13 9PT; and Centre for Genome Research (A.G.S.. I.C.), University of Edinburgh, Edinburgh EH9 3QJ, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. J. R. A. Sherwin, Department of Obstetrics and Gynaecology, University of Cambridge, Box 223, The Rosie Hospital, Cambridge CB2 2SW, United Kingdom. E-mail: jras100{at}cam.ac.uk.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We used a mouse model to identify genes that play an important role in the acquisition of receptivity. In the mouse, the uterus does not become fully receptive to the attaching embryo until exposed to nidatory estrogen, on the morning of d 4 of pregnancy (7). Implantation begins on the evening of d 4 of pregnancy, approximately 92 h post coitus (pc), and by midday of d 5 the blastocyst can no longer be easily flushed from the uterine cavity (8). The first sign of attachment is increased vascular permeability at the site of implantation. The endometrium responds to the embryo by undergoing decidualization, characterized by local proliferation and differentiation of endometrial stromal fibroblasts. Embryo transfer experiments have shown that the uterus remains receptive to the implanting embryo for approximately 24 h between d 4 and d 5 of pregnancy (2). The mechanism by which nidatory estrogen brings the endometrium to a fully receptive state is not understood. However, on d 4 of pregnancy, the nidatory estrogen surge induces a transient rise in expression of the cytokine LIF in endometrial glandular epithelium (9). This LIF expression is essential for implantation, because animals lacking the LIF gene (LIF–/–) produce normal blastocysts that fail to implant in the LIF-deficient uterus but are capable of implantation in a wild-type uterus (3). Intraperitoneal or intrauterine injection of recombinant LIF on d 4 of pregnancy to LIF–/– animals restores implantation (3, 10). The uterine expression of LIF is independent of the presence of an embryo, and females heterozygous for the LIF gene (LIF–/+) show normal up-regulation of LIF expression on d 4 of pregnancy or pseudopregnancy and are fertile. Taken together, these results indicate that LIF synthesized in the glandular epithelium, in response to the nidatory estrogen surge, primes the endometrium for blastocyst attachment, by signaling through the LIF receptor, localized to the LE cells (11, 12).
There is strong evidence that LIF is involved in implantation in several other species (reviewed in Ref. 13). In many cases LIF expression in the endometrium increases at the time of blastocyst implantation. Intrauterine injection of neutralizing antibody against LIF significantly reduces implantation rates in rhesus monkeys, and in sheep passive immunization against LIF also reduces pregnancy rates (14, 15). In women, the concentration of LIF in endometrial fluid from patients with unexplained infertility is reduced compared with normal fertile controls, but an essential role for LIF in human implantation remains unproven (16). However, the mechanism by which the action of LIF, in conjunction with other effects of steroids, renders the endometrium receptive has not been determined.
In this study we sought to identify genes regulated by LIF in mouse endometrium at the time of implantation. One horn of the uterus of each LIF-deficient mouse was injected with recombinant LIF and the other was injected with PBS on d 4 of pseudopregnancy. After comparison of the mRNA populations from the LIF- and PBS- treated horns using Affymetrix oligonucleotide arrays, we identified IGF-binding protein 3 (IGFBP3) as a gene up-regulated by LIF treatment. In situ hybridization showed that the primary site of IGFBP3 expression is the LE, the known site of action of LIF in the uterus at this time. Two further LIF-regulated genes, Amphiregulin (Ar) and immune response gene 1 (IRG1) were also identified. Expression of all three of these genes has previously been shown to be regulated by P4. This study has therefore identified three genes that are expressed in the LE at implantation under the combined regulation of LIF and P4.
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Identification of LIF-Responsive Genes by Microarray Analysis
To identify genes regulated by LIF in the mouse uterus, the left uterine horn of pseudopregnant LIF–/– mice (n = 4) was injected with 400 ng recombinant human LIF at 84 h of pseudopregnancy (afternoon of d 4). The right horn was injected with PBS. Total RNA was isolated 12 h later from each uterine horn and analyzed using Affymetrix oligonucleotide microarrays. The transcript abundance for each cDNA represented on the array was compared between LIF- and PBS-treated horns from the same animal using Affymetrix GeneChip Microarray Suite version 5.0 software. This determines whether the hybridization signals for each transcript are significantly different between the paired samples and classifies them as decreased, increased, or unchanged. A gene was accepted as significantly different if it was classified as increased or decreased by more than 1.5-fold in at least three of the four animals. The transcript IGFBP3 was the only transcript that fulfilled these criteria and which also had an expression level greater than three SDs above the background. Real-time RT-PCR was used to verify the findings from the Affymetrix gene arrays using primers and probe specific for murine IGFBP3 (Table 1
). cDNA samples from an additional 10 paired uterine horns from LIF–/– animals that had been treated identically to those used in the Affymetrix gene array comparisons were generated. The level of IGFBP3 cDNA in each sample was measured relative to a reference RNA, and the values were corrected for differences in loading relative to the 18S rRNA. In nine of 10 animals, LIF treatment increased the level of the IGFBP3 transcript compared with the paired PBS-treated horn (Fig. 1). The mean fold increase in the LIF-treated horns was 2.3-fold (P = 0.02).
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). Total uterine RNA was isolated at 96 h during pseudopregnancy. This is some 18 h after the transient peak of endogenous LIF expression in the uterus, which is reported to occur on the morning of d 4 pc (between 72 and 80 h pc) (9, 17). IGFBP3 expression was significantly reduced in LIF–/– mice (0.64 ± 0.04, mean ± SEM), compared with LIF+/– littermates (1.0 ± 0.12, P = 0.045). This supports the view that IGFBP3 is up-regulated by LIF in the normal periimplantation uterus.
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90 h in pregnancy). Total RNA was isolated from normal MF1 pseudopregnant animals between 84 and 120 h pc. The level of IGFBP3 expression was determined by real-time RT-PCR relative to an arbitrary reference sample of total uterine RNA. IGFBP3 showed a dramatic increase in overall expression level during this time with median levels of expression of 0.49. 1.0, 2.0, and 4.4 at 84, 96, 108, and 120 h, respectively (Fig. 3). The level of IGFBP3 begins to increase between 84 and 96 h, shortly after the expected peak of LIF expression.
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). There was strong and distinct hybridization of the IGFBP3 antisense riboprobe to LE with some weak, diffuse stromal expression (see Fig. 4, A and B). This hybridization to the LE appeared to show a gradient, being stronger on the antimesometrial side, the site of blastocyst attachment in normal pregnancy. No specific hybridization was observed with the sense probe on adjacent sections (Fig. 4C). At earlier stages of pseudopregnancy, the expression pattern was similar, although the overall intensity of hybridization was greatly decreased (data not shown). The site of IGFBP3 expression was also examined during pregnancy in wild-type mice. It was identical to that seen up to 96 h of pseudopregnancy (data not shown). However, by 120 h of pregnancy, implantation has occurred in normal mice. Figure 4, D–F, shows the implantation chamber at 120 h pc. There is strong expression of IGFBP3 transcripts in the primary zone of decidualization immediately surrounding the implanting embryo as well as in the LE. Hybridization of the antisense probe is also seen in some, but not all, glandular epithelium.
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). Expression of IRG1 mRNA, which is known to be regulated by P4 were also measured to confirm the mice had responded to P4 as expected. IGFBP3 mRNA was decreased by E2 treatment at both 6 and 24 h. P4 injection also dramatically decreased IGFBP3, but this effect was only seen 24 h post injection. Treatment with E2 and P4 together also greatly decreased IGFBP3 expression. In contrast, IRG1, which is also expressed in the LE, was strongly induced by P4 at 6 h and 24 h, and by E2 and P4 together with no effect seen by E2 alone, as expected (18, 19).
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). These were the same samples as used in Fig. 1
(n = 10). Amphiregulin expression showed a mean increase of 7.2-fold, and IRG1 increased an average of 3.7-fold in response to LIF injection compared with the PBS-treated horn from the same animal (P < 0.002 in both cases). In contrast, there was no apparent effect of intrauterine LIF injection on the expression of the two other P4-responsive genes examined, OSF2 and L-12/15-LOX (data not shown).
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DISCUSSION |
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In this study we used oligonucleotide microarrays to compare the transcript profiles of paired uterine horns from LIF-deficient MF1 mice, 12 h after intraluminal injection of LIF or PBS on d 4 of pseudopregnancy. LIF injection restored implantation in these animals, and d 4 corresponds to the normal time of uterine LIF expression in pregnancy and pseudopregnancy (9). The use of paired uterine horns from the same animal ensured that the LIF- and PBS (control)-treated endometriums were exposed to identical hormonal environments. IGFBP3 was shown to increase in response to LIF injection, and this was confirmed by real-time RT-PCR in independent experiments. We also compared IGFBP3 expression at 96 h of pseudopregnancy in LIF–/– and LIF+/– littermates. This is approximately 18 h after the normal peak of LIF expression induced by nidatory E2 (9) and corresponds to the receptive period induced by LIF. Real-time RT-PCR showed that IGFBP3 expression was reduced in the LIF–/– mice, supporting the view that IGFBP3 is up-regulated by LIF in vivo. However, IGFBP3 expression was not absent in LIF–/– animals, indicating that its expression in the uterus is not solely dependent upon LIF. A recent microarray study has shown that administration of RU486 on d 3 of pregnancy results in reduced expression of IGFBP3 on d 4 (18). RU486 is primarily known as an antiprogestin; however, it can exhibit agonist activity and also has antiglucocorticoid and antiandrogen actions (22, 23). Although the effect of RU486 on IGFBP3 was not verified by an independent method, it strongly suggests a role for P4 in the regulation of IGFBP3 expression. Surprisingly, we showed that acute administration of E2 or P4 to ovariectomized animals results in dramatic down-regulation of IGFBP3. A similar down-regulation was seen with combined E2+P4 treatment. However the response of IGFBP3 on d 4 of pregnancy to P4 after several days of rising E2 may be different from that seen in ovariectomized animals.
In situ hybridization showed that at 96 h of pseudopregnancy, IGFBP3 mRNA is primarily expressed in the LE, in wild-type mice. This is the expected site for genes directly regulated by LIF in mouse uterus, as the functional LIF receptor complex is expressed in the same site (11, 12). However, we found some IGFBP3 expression in LE on d 3 of pregnancy, before endogenous LIF expression on d 4. This may represent IGFBP3 expression due to the action of the P4 receptor (PR), which is localized to the epithelium at this time (18, 24). IGFBP3 mRNA expression was also detected in the stroma at 120 h of pregnancy, around the implanting blastocyst in decidualizing stromal cells. Taken together, these data suggest that IGFBP3 expression in the uterus may be regulated by the combined action of LIF and P4. The relatively low expression in LE on d 3 of pregnancy or pseudopregnancy may be as a result of the actions of P4, whereas on d 4, both LIF and P4 may be required together to achieve the high levels of IGFBP3 expression seen in LE. In the absence of LIF, IGFBP3 expression is reduced as we found in Fig. 2. After implantation, novel IGFBP3 expression in the stroma coincides with the process of decidualization and may be mediated by local embryo-derived factors. In humans, coculture of endometrial stromal cells with preimplantation embryos stimulates IGFBP3 production (25). IGFBP3 is a member of the IGF family, which consists of two growth factors (IGF-I and IGF-II), two receptors (IGF-IR and IGF-II/mannose-6-phosphate receptor), and six binding proteins (IGFBPs 1–6) (for reviews see Refs. 26 and 27). The IGFs exhibit mitogenic and antiapoptotic effects, whereas the IGFBPs modulate IGF bioavailability, but also have IGF-independent actions in tissue. IGFs and IGFBPs have been shown to stimulate metabolism and cell proliferation in preimplantation mouse embryos (28). However, no significant change in fecundity or litter size was noted in IGFBP3 null mice (29, 30). Therefore, IGFBP3 does not appear to be essential for the acquisition of endometrial receptivity mediated by LIF. This may be due to the functional redundancy that exists within the IGFBP family, because the six IGFBPs exhibit many similar properties. Localization of IGFBP3 expression in mouse uterus has previously been attempted using immunohistochemistry (31). Diffuse stromal and decidual immunostaining was seen in early pregnancy, but no detailed analysis of periimplantation LE was made. In situ hybridization studies of IGFBP3 expression in rat uterus confirm the up-regulation of IGFBP3 expression in LE in this species at the time of implantation (32).
We have shown that the expression of two further genes, amphiregulin and IRG1, are up-regulated by LIF at the time of implantation. These genes are part of a group that have an expression profile similar to LIF, being transiently expressed in uterine LE on d 4 of pregnancy (20). Other genes in this group include OSF2, L-12/15-LOX, and Fisp12. All of these genes have been shown to be up-regulated at the time of implantation by the action of P4, and their expression is reduced by the antiprogestin RU486 (20). We found no effect of LIF injection on the expression of OSF2 and L-12/15- LOX on d 4 of pseudopregnancy in LIF-deficient mice. However, intrauterine LIF injection up-regulated expression of amphiregulin and IRG1 (7.2- and 3.7-fold, respectively) in LIF-deficient mice. Although these two genes are clearly regulated by LIF, they were not identified in our microarray experiment. The expression levels, as determined by the microarrays, were close to background. This illustrates the limitations of hybridizing total uterine RNA to microarrays to analyze gene expression changes induced by LIF in LE, which comprises less than 5% of the uterus. An alternative approach is to isolate RNA from purified LE before array analysis. These experiments are underway.
Amphiregulin is a member of the epidermal growth factor (EGF) family, which includes EGF, heparin-binding EGF (HB-EGF), betacellulin, epiregulin, and TGF-
. They are synthesized as transmembrane proteins, which can be cleaved to produce soluble forms. Several members of the family appear to be involved in implantation. HB-EGF, betacellulin, and epiregulin are locally up-regulated in LE and stroma underlying the implanting blastocyst, probably in response to embryo-derived signals (33). In contrast, amphiregulin is considered a marker of receptive endometrium, because its expression throughout the LE is initiated on d 4 of pregnancy, before implantation, in response to P4 (34). The expression of amphiregulin, HB-EGF, and epiregulin have previously been examined by in situ hybridization on d 4 of pregnancy in LIF–/– mice. HB-EGF and epiregulin, which are normally expressed in epithelium beneath the blastocyst, were not expressed in LIF–/– animals (35). This probably reflects the failure to initiate endometrial responses to the embryo in these animals. In situ hybridization studies of amphiregulin expression on d 4 of pregnancy showed reduced expression in the LE of LIF–/– animals. Reduced up-regulation of amphiregulin in response to P4 injection of ovariectomized LIF–/– mice was also reported; however, these responses were not quantified. We have now shown that amphiregulin expression is increased 7-fold in LIF–/– mice in direct response to intrauterine injection of LIF. However, amphiregulin is not essential for implantation, because mice simultaneously deficient for EGF, TFG-, and amphiregulin are fertile (36). Therefore, amphiregulin expression does not appear to be essential for the LIF-mediated acquisition of endometrial receptivity. As with IGFBP3, this may be due to redundancy within the EGF family.
The full-length IRG1 cDNA encodes a protein of 488 amino acids (19). IRG1 is expressed in LE with peak expression coinciding with implantation on d 4 of pregnancy (18). This expression is primarily regulated by P4, because IRG1 expression in the mouse uterus on d 4 is reduced 8-fold by prior treatment with the antiprogestin RU486, and mice deficient for progesterone receptor (PR) fail to express IRG1 (20). However, this same study reported that full physiological expression was shown to require the action of the nidatory E2 surge (19). The idea that full IRG1 expression at the time of implantation requires the synergistic action of P4 and nidatory E2 is supported by our findings, because coadministration of E2 and P4 to ovariectomized mice produced significantly higher IRG1 expression at 24 h compared with P4 alone. In addition, our results indicate that the effect of E2 on IRG1 is mediated by LIF. We have shown that intrauterine LIF injection on d 4 of pregnancy into LIF–/– mice increases IRG1 expression by 3.7-fold. In contrast, Chen et al. (19) also reported no difference in IRG1 expression between LIF–/– and LIF+/– of animals on d 4 of pregnancy. The reason for this discrepancy is unclear. One explanation may be in the timing of sample collection. Given that the peak of LIF expression occurs in the morning of d 4, it is probable that the up-regulation of IRG1 that we see 12 h after intrauterine LIF injection may not be evident if LIF–/– and LIF+/– animals are compared too early on d 4. The identification of IRG1 as a LIF-regulated gene is important because IRG1 has recently been shown to play an essential role in implantation. Treatment with antisense oligodeoxynucleotides to IRG1 on d 3 of pregnancy decreased its expression on d 4 and reduced implantation by approximately 80% (18). IRG1 is highly conserved in vertebrates and strongly resembles the bacterial enzyme methylcitrate dehydratase. The enzyme activity of the mammalian ortholog has not been tested, but Chen et al. (19) propose that IRG1 may play a role in odd-chain lipid metabolism. This may explain the effect of IRG1 on implantation, because several other enzymes that affect fatty acid metabolism are also known to be important in implantation. Leukocyte 12/15 lipoxygenase activity has been shown to be essential for implantation, although in this study we have shown that this lipoxygenase is not regulated by LIF (37). Similarly, mice deficient in cyclooxygenase 2, which is involved in prostaglandin synthesis, exhibit delayed or reduced implantation (38, 39). Because our data indicate that IRG1 is regulated by LIF at implantation, we would predict that fatty acid metabolism would be altered in the uteri of LIF–/– mice on d 4 of pregnancy and pseudopregnancy. Experiments to test this hypothesis are underway.
Possible explanations for the decreased expression of P4-responsive genes in LIF–/– animals include reduced systemic P4 levels or altered PR expression in the endometrium. However plasma P4 levels and PR expression are apparently normal in LIF–/– animals (10, 35). Moreover, this study and others have shown that many P4-regulated genes such as OSF-2 are normally expressed in LIF–/– animals (10, 35). Therefore, the reduced expression of IGFBP3, amphiregulin, and IRG1 that we have found in these animals is not due to a global alteration in P4 responses. LIF signaling through the LIFR/gp130 receptor complex, results in phosphorylation of the signal transducers and activators of signaling (STATs) which translocate to the nucleus, where they regulate gene transcription (40). The effect of LIF on LE, isolated on d 4 of pregnancy, is phosphorylation of STAT-3, with no increase in MAPK activity (12). The idea that the STAT-3 signal transduction pathway is important in the acquisition of receptivity is confirmed by gp130
STAT transgenic mice. These animals have a gp130 receptor, which is deleted for STAT binding sites, although the receptor is still competent for SHP-2/ras/Erk activation (41). The absence of STAT-dependent responses results in a phenotype identical to the LIF-deficient animals, with failure of blastocyst implantation. If LIF signaling through STATs is critical for its effects on implantation, how might coregulation of genes such as IRG1 and IGFBP3 by LIF and P4 occur? The putative promoter regions of IGFBP3, IRG1, and amphiregulin were interrogated for candidate STAT-3 response elements and P4 response elements (using online software at http://www.gene-regulation.com/. No STAT-3 response elements were identified in the promoter regions of these genes, although P4 response elements were readily identified upstream of IGFBP3 and amphiregulin. This suggests that they may not be regulated directly by STAT-3. An alternative mechanism has been suggested by the finding in rat decidua that phosphorylated STAT-3 can bind to the PR complex and so may regulate its activity (42). Immunohistochemistry shows PR expression in LE and stroma on d 3 of pregnancy, but PR expression declines markedly in LE on d 4, although it remains detectable (18, 24). STAT-3 and the LIFR complex are expressed in LE on d 3–5 of pregnancy, so PR and functional LIFR/STAT-3 may act in synergy to coregulate expression of IGFBP3, amphiregulin, IRG1, and other genes expressed in LE. Alternatively, P4 may regulate gene expression in the LE through a paracrine mechanism, in which P4 acts on the stroma to induce local factors, which then act on the LE.
Embryo transfer experiments in many species have confirmed the concept that the endometrium becomes receptive to the embryo for a limited time after ovulation. It is likely that this period of uterine receptivity is characterized by a particular pattern of gene expression, which renders the endometrium responsive to embryonic signals. Recently, several genes, which appear to be essential for normal implantation, in addition to the PR, have been identified in rodents. These include LIF, L-12/15-LOX, hmx3, calcitonin, and IRG1. However, little is known of the mechanisms by which these genes contribute to uterine receptivity. To date, only one gene, coch-5b2, has been shown to be regulated directly by LIF in mouse uterus at the time of implantation (43). Mice deleted for the coch gene show a normal phenotype, suggesting that coch is not essential for reproduction in mice. This study has identified three further genes, IGFBP3, amphiregulin, and IRG1, that are regulated by LIF in the uterus. IRG1 has recently been shown to play an essential role in implantation and may thus be a major mediator of the downstream actions of LIF. In contrast, L-12/15-LOX, is not LIF regulated and may carry out a different function essential for implantation. This study suggests that the normal physiological expression of IGFBP3, amphiregulin, and IRG1 in the mouse uterus at implantation requires the actions of both LIF and P4. The possibility of the coregulation of gene expression by LIF and P4 provides a new concept in the understanding of the mechanism by which the endometrium is brought to a receptive state. Further studies of the expression and function of genes such as IRG1 will be required to determine whether they are involved in the acquisition of uterine receptivity in other species including humans.
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Tissue Preparation and RNA Isolation
Pseudopregnant animals were obtained by placing females with T145C males together at 2400 h. The animals were separated 4 h later and checked for vaginal plugs. This time was designated t = 0 h of pregnancy. Uterine samples were prepared by dissection at timed intervals from 72–120 h of pregnancy. The uterine horns were cut into 2-mm pieces. One piece was fixed in 10% neutral buffered formalin, for 6 h at room temperature and then processed through graded ethanol and paraffin embedded for analysis by in situ hybridization. The majority of the uterus was snap frozen in liquid nitrogen, and stored at –70 C before RNA extraction. Total RNA was extracted by homogenization in 3 ml of TRIzol (Invitrogen, Paisley, Scotland, UK), according to the manufacturer’s protocol.
Affymetrix Oligonucleotide Microarray and Data Analysis
To generate uterine RNA for microarray analysis, 400 ng recombinant human LIF (R&D Systems, Minneapolis, MN) was injected in a volume of 10 µl PBS containing 0.1% BSA into the lumen of one uterine horn at 84 h of pseudopregnancy in LIF–/– females. The contralateral horn was injected with 10 µl PBS/BSA. The animals were killed 12 h later (96 h pc), and samples of uterus were fixed for in situ hybridization or frozen for RNA isolation. Total RNA was isolated as described above. Total RNA isolated from paired control and LIF-treated uterine horns from individual mice was subjected to expression analysis using Affymetrix mouse MG-U74Av2 oligonucleotide microarrays (Affymetrix, Santa Clara, CA). These microarrays contain oligonucleotides corresponding to approximately 12,000 known mouse genes and unnamed expressed sequence tags. A full gene listing is available at: http://www.affymetrix.com/products/arrays/specific/mgu74.affx. The total RNA was further purified using RNeasy columns (QIAGEN, Crawley, UK), and then cDNA from each horn was prepared according to the protocol recommended by Affymetrix Inc. Total RNA (10 µg) was reverse transcribed with a T7 oligo-dT primer using the Superscript Choice system (Invitrogen, San Diego, CA). In vitro transcription was performed using the ENZO Bioarray high yield RNA T7 labeling kit (Enzo, Farmingdale, NY). The resulting cRNA was cleaned up using RNeasy spin columns (QIAGEN) and fragmented for 35 min at 94 C in 40 mM Tris-acetate, pH 8.1; 100 mM KOAc; 30 mM MgOAc. Hybridization to mouse MG-U74Av2 arrays and laser scanning was carried out at the Human Genome Mapping Project (HGMP) center at Hinxton Hall Cambridge using a GeneChip Fluidics Station 400 and an Agilent GeneArray scanner. Scanned images were analyzed using the Affymetrix GeneChip Microarray Suite (version 5.0) and scaled transcript abundance data for each pair of uterine horns were compared. The Microarray Suite (version 5.0) software uses a published alogarithm (http://www.affymetrix.com/products/software/index.affx) to determine whether the signal for each transcript differs significantly between the pair of samples and classifies them as decreased, increased, or no change. A gene was accepted as significantly different if it was classified as increased or decreased by more than 1.5-fold in at least three of the four animals.
Real Time RT-PCR
To verify the results from the microarray analysis, real-time RT-PCR was performed for IGFBP3 mRNA (X81581) using the ABI PRISM 7700 sequence detection system (Taqman, Applied Biosystems, Warrington, UK). Primers and probe were designed using Primer Express v1.5 software (Applied Biosystems). The probes were labeled with 5'-FAM and 3'-TAMRA (Oswell, Southampton, UK). Primers were also designed to measure the expression of mouse mRNAs encoding IRG1 (L38281) and OSF2 (NM_015784). The sequences are shown in Table 1. Presynthesized primers and probe sets were used to determine the relative levels of mRNAs encoding mouse amphiregulin and Leukocyte-12/15-LOX (product nos. Mm00437583 and Mm00507789, Assays-on-demand, Applied Biosystems). The endogenous control 18S rRNA was assayed using primers and probes supplied by Applied Biosystems. Probe and primer optimization and real-time PCR were performed using the manufacturer’s recommended conditions. Standard curves were generated by serial dilution of a standard preparation of total RNA isolated from d 8 pregnant mouse uterus. Data are expressed in arbitrary units relative to the level of the same gene in this standard RNA. cDNA was produced from each sample of uterus by reverse transcription using 3 µg total RNA with 200 IU Superscript RT and random primers (Invitrogen) according to the manufacturer’s instructions. The expression values obtained were normalized against those from the control ribosomal 18S mRNA to account for differing amounts of starting material.
Statistical Methods
Gene expression levels in the LIF and control treated horns, derived from RT-PCR data, were compared using the nonparametric Mann-Whitney test for paired samples. Differences were considered statistically significant when P < 0.05.
Radioactive In situ Hybridization
[33P]UTP-labeled single-stranded sense and antisense riboprobes were synthesized from 1 µg linearized IGFBP3 cDNA template using a MAXIscript in vitro transcription kit (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. The probe corresponded to 328 bp between bases 572 and 900 of mouse IGFBP3 (X181581). The sense and antisense probes were hybridized to formalin-fixed sections of mouse uterus as described previously with minor modifications (44). After deparaffinization and rehydration, sections were pretreated with 0.2 M HCl for 20 min, and then 2x sodium chloride/sodium citrate buffer (SSC) for 30 min, at room temperature. The sections were digested for 20 min at 37 C with proteinase K at 2 µg/ml in 0.2 M Tris (pH 7.2), 0.05 M EDTA (pH 8.0). Two treatments of 5% (vol/vol) acetic anhydride in 100 mM triethanolamine were performed for 5 min each, before sections were rinsed in 2x SSC. Sections were hybridized with 50 µl hybridization solution containing 40,000 cpm/µl of riboprobe for 18 h at 50 C under a coverslip. After hybridization, the sections were washed in two changes of 5x SSC at 50 C over 30 min, and then 2x SSC, 50% (vol/vol) formamide for 30 min at 65 C, before being rinsed in four washes of 2x SSC at 37 C over 20 min. After these washes the slides were incubated in 400 mM NaCl, 0.1 M Tris-HCl (pH 7.5), 0.05 M EDTA (pH 8.0) containing 20 µg/ml RNase A (Sigma Chemical Co., St. Louis, MO) at 37 C for 30 min. The slides were then washed in 2x SSC, 50% (vol/vol) formamide solution for 30 min at 65 C, followed by 2x SSC and 0.2x SSC both for 15 min at 37 C. The sections were dehydrated through an ethanol series [30%, 60%, 80%, and 95% (vol/vol) in 0.3 M ammonium acetate, two rinses in 100% ethanol] and air-dried. The slides were dipped in LMI emulsion (Amersham Pharmacia Biotech, Arlington Heights, IL) and developed after 14–21 d using Kodak D19 developer (Eastman Kodak, Rochester, NY). Developed sections were counterstained for 10 sec using Gills hematoxylin (Sigma) and then dehydrated and mounted in Depex (British Drug House Ltd., Poole, UK).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: E2, 17ß-Estradiol; EGF, epidermal growth factor; HB-EGF, heparin-binding EGF; IGFBP3, IGF-binding protein 3; IRG1, immune response gene-1; LE, luminal epithelium; LIF, leukemia-inhibitory factor; LIFR, LIF receptor; L-12/15-LOX, leukocyte 12/15 lipoxygenase; OSF2, osteoblast-specific factor 2; P4, progesterone; pc, post coitus; PR, progesterone receptor; SSC, sodium chloride/sodium citrate; STAT-3, signal transducer and activator of transcription-3.
Received for publication March 16, 2004. Accepted for publication May 25, 2004.
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REFERENCES |
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regulates implantation in mice. J Biol Chem 279:11570–11581 or ß A inhibin subunit mRNA is expressed in ovine fetal ovaries in late gestation. J Mol Endocrinol 13:1–9This article has been cited by other articles:
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  L Mohamet, J K Heath, and S J Kimber Determining the LIF-sensitive period for implantation using a LIF-receptor antagonist Reproduction, November 1, 2009; 138(5): 827 - 836. [Abstract] [Full Text] [PDF]
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C. Ponce, M. Torres, C. Galleguillos, H. Sovino, M A. Boric, A. Fuentes, and M C. Johnson Nuclear factor {kappa}B pathway and interleukin-6 are affected in eutopic endometrium of women with endometriosis Reproduction, April 1, 2009; 137(4): 727 - 737. [Abstract] [Full Text] [PDF]
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 A.A Fouladi-Nashta, L Mohamet, J.K Heath, and S.J Kimber Interleukin 1 Signaling Is Regulated by Leukemia Inhibitory Factor (LIF) and Is Aberrant in Lif-/- Mouse Uterus Biol Reprod, July 1, 2008; 79(1): 142 - 153. [Abstract] [Full Text] [PDF]
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 L. Aghajanova, K. Bjuresten, S. Altmae, B.-M. Landgren, and A. Stavreus-Evers HB-EGF but Not Amphiregulin or Their Receptors HER1 and HER4 Is Altered in Endometrium of Women With Unexplained Infertility Reproductive Sciences, May 1, 2008; 15(5): 484 - 492. [Abstract] [PDF]
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S J Kimber, S F Sneddon, D J Bloor, A M El-Bareg, J A Hawkhead, A D Metcalfe, F D Houghton, H J Leese, A Rutherford, B A Lieberman, et al. Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors Reproduction, May 1, 2008; 135(5): 635 - 647. [Abstract] [Full Text] [PDF]
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 W. V. Ingman and R. L. Jones Cytokine knockouts in reproduction: the use of gene ablation to dissect roles of cytokines in reproductive biology Hum. Reprod. Update, March 1, 2008; 14(2): 179 - 192. [Abstract] [Full Text] [PDF]
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A. A Gratao, M. Dahlhoff, F. Sinowatz, E. Wolf, and M. R Schneider Betacellulin Overexpression in the Mouse Ovary Leads to MAPK3/MAPK1 Hyperactivation and Reduces Litter Size by Impairing Fertilization Biol Reprod, January 1, 2008; 78(1): 43 - 52. [Abstract] [Full Text] [PDF]
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 E.A. Campbell, L. O'Hara, R.D. Catalano, A.M. Sharkey, T.C. Freeman, and M. H. Johnson Temporal expression profiling of the uterine luminal epithelium of the pseudo-pregnant mouse suggests receptivity to the fertilized egg is associated with complex transcriptional changes Hum. Reprod., October 1, 2006; 21(10): 2495 - 2513. [Abstract] [Full Text] [PDF]
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E. Dimitriadis, C.A. White, R.L. Jones, and L.A. Salamonsen Cytokines, chemokines and growth factors in endometrium related to implantation Hum. Reprod. Update, November 1, 2005; 11(6): 613 - 630. [Abstract] [Full Text] [PDF]
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S. J Kimber Leukaemia inhibitory factor in implantation and uterine biology Reproduction, August 1, 2005; 130(2): 131 - 145. [Abstract] [Full Text] [PDF]
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 R. D. Catalano, M. H. Johnson, E. A. Campbell, D. S. Charnock-Jones, S. K. Smith, and A. M. Sharkey Inhibition of Stat3 activation in the endometrium prevents implantation: A nonsteroidal approach to contraception PNAS, June 14, 2005; 102(24): 8585 - 8590. [Abstract] [Full Text] [PDF]
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G. Schofield and S. J. Kimber Leukocyte Subpopulations in the Uteri of Leukemia Inhibitory Factor Knockout Mice During Early Pregnancy Biol Reprod, April 1, 2005; 72(4): 872 - 878. [Abstract] [Full Text] [PDF]
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F. L Lopes, J. A Desmarais, and B. D Murphy Embryonic diapause and its regulation Reproduction, December 1, 2004; 128(6): 669 - 678. [Abstract] [Full Text] [PDF]
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